Supplemental Methods - Young Scientist Journal

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12 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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SUPPLEMENTAL METHODS

Site
-
directed

Mutagenesis

Once candidate
H. pylori

DAF
-
b
inding proteins were identified,

alpA, alpB
, and
hofC

mutants were generated. Target genes
of the alpA proteins were mutagenized by gene splicing by overlap extension (gene
SOEing), which allows for genetic
engineering of novel restriction sites for directional cloning of chloramphenicol or kanamycin
-
encoding resistance
cassettes within specified regions of the
H. pylori
chromosome. In the primary PCR step, two reactions were

performed to
amplify the upstream and downstream portions of the gene of interest. Secondary PCR was then
performed

for
alpA

to
generate DNA with novel BamHI sites. The
alpB

and
hofC

genes both contained the restriction site BamHI so only
amplification

of the genes was necessary. The final PCR products were gel extracted, purified (Qiagen), and cloned into
the pGEM
-
T Easy vector (Promega). Kanamycin or chloramphenicol
-
encoding resistance cassettes were inserted into the
genes via the BamHI sites, and th
e plasmids were transformed into
H. pylori
.
flaA
-

and
flaB
-

isogenic mutants and
the
hopQ
-

strain 26695 mutants provided by different labs were subjected to primary PCR as well as additional PCR to detect
kanamycin or chloramphenicol
-
encoding resistance ca
ssettes.
M
utants were tested for resistance on kanamycin and
chloramphenicol plates, and the chromosomal DNA from
flaA
-
, flaB
-
,

and
hopQ
-

were used to transform
H. pylori

strains
J166 and 7.13.
H. pylori

mutants were grown on plates supplemented with eithe
r chloramphenicol or kanamycin for
selection of positive transformants.


Liquid

Chromatography
-
Mass

Spectrometry

(
LC
-
MS
)
.

Bands

separated by SDS
-
PAGE were analyzed to determine proteins of interest. Bands that appeared in the lanes with
H.
pylori

proteins
and DAF, but not in the lanes with
H. pylori

proteins alone were extracted from the silver stained gel,
destained, and sent to the Vanderbilt Proteomics Laboratory for in
-
gel digestion with trypsin protease. The peptides were
extracted and analyzed by reve
rse
-
phase liquid chromatography coupled in
-
line with electrospray ionization and tandem
mass spectrometry, which was performed using a ThermoScientific LTQ linear ion trap mass spectrometer. Electrospray
ionization of peptides was performed using a Thermo
nanospray source modified for automated vented column injection.
Resulting peptide MS/MS fragmentation spectra were analyzed with the Sequent algorithm to produce candidate protein
identifications based on correlation of fragmentation patterns with predict
ed patterns based on protein sequence
databases. Filtering of Sequest matches was done in the lab according to percentage of total spectra, correlation of gel
region with predicted molecular weight, and cellular role.